It has been known to determine cholesterol in sera by the use of assay compositions based on cholesterol oxidase, presently from a microbial source. The reaction involved is: ##STR1##
For total cholesterol determination, bound cholesterol may be released by the inclusion of cholesterol esterase which yields cholesterol by the reaction: ##STR2##
The amount of cholesterol can be assayed by measuring the amount of oxygen consumed, the amount of cholest-4-en-3-one formed, or the amount of hydrogen peroxide formed. A preferred way is to determine the amount of hydrogen peroxide formed by use of a chromogen system. A preferred chromogen system is one based on the presence of peroxidase from a horseradish source, phenol and antipyrine involving the reaction: ##STR3##
While most assay systems based on cholesterol oxidase can be made functional as prepared, they are prone to rapid degradation. As a consequence, the art early on lyophilized (freeze-dried) the composition for reconstitution at the time of use. Liapholization is expensive and suffers from inaccuracy.
A need was recognized to provide a liquid assay system of controlled composition which would have an adequate shelf life for marketing purposes. As invented and described by one of us, and disclosed in EPC Application 80.104.568.3, filed Aug. 1, 1980, incorporated herein by reference, it was found that the presence of a material quantity, e.g., up to 50 percent by volume, of a polyhydroxy compound such as glycerol would induce long shelf life to a liquid assay composition. The invention enabled precise quality control to be exercised over the composition of the system, and enabled total reliability of the assay system as a tool. The system was formulated as a concentrate. Shelf life of the concentrate was more than adequate for industrial use and provided levels of stability theretofore unknown in the art.
The polyhydroxy compound, while functional to stabilize the system against degradation, increases costs and, unless proper housekeeping procedures are followed, contaminates apparatus, affecting other tests, particularly triglyceride analysis.
A desire has existed, therefore, for a liquid assay system which did not yield in performance, which could be sold as a single formulation for use as is without dilution and yet have an adequate shelf life to satisfy marketing requirements.